The immune system is used to combat bacteria, viruses, and foreign multicellular organisms, as well as cancerous cells. Immune responses are provided by cells of the bone marrow, spleen, and other tissues. Unfortunately, improper regulation of the immune system can result in a number of disorders or pathological conditions. These disorders or conditions include chronic inflammation, autoimmune disease, and undesired allergic reactions to foreign particles or foreign tissues.
Cells of the immune system possess many types of membrane-bound proteins that serve as receptors. The ligands for these receptors may be small molecules, proteins, e.g., cytokines or chemokines, or membrane-bound proteins residing on a separate cell. The occupation of a receptor by its ligand, binding of a receptor by a soluble antibody, cross-linking of like-receptors to each other, and cross-linking of unlike receptors to each other, can result in changes in cellular activity. Some of these events result in “cell activation,” while other events result in “cell inhibition.”
Studies of immune cells and their activation or inhibition have related to: Recruitment of enzymes to the plasma membrane; recruitment of enzymes to “lipid rafts” in the cell membrane (Yang and Reinherz, J. Biol. Chem. 2766, 18775 (2001)), and recruitment of membrane-bound receptors to the plasma membrane. A lipid raft is a region of the plasma membrane with reduced fluidity of the lipid molecules. Cell activation or inhibition also relates to changes in phosphorylation state of receptors; changes in the proliferative state of the cell; calcium fluxes; changes in genetic expression; changes in secretion or in degranulation; differentiation of the cell; changes in the proliferative rate of the cell; changes in cell migration; and changes in chemotaxis. Cell activation may also include the reversal of T cell anergy (see, e.g., Lin, et al., J. Biol. Chem. 273, 19914 (1998); and Sunder-Plassman and Reinherz, J. Biol. Chem. 273, 24249 (1998)).
The question of whether a signaling event, which results in any of the above changes, is activating or inhibiting can be determined on an individual basis. For example, if occupation of an unidentified receptor results in an increases of genetic expression of cytokine mRNA, secretion (or degranulation), release of inflammatory cytokines, phagocytic or lytic activity, the unidentified receptor may be termed an activating receptor. Similarly, if occupation of an unidentified receptor inhibits activity dependent on a known activating receptor, then that unidentified receptor may be termed an inhibiting receptor.
The determination of whether a receptor is activating or inhibiting may be predicted by the polypeptide sequence of the receptor, where the receptor is a protein. Attention has focused on two different motifs: ITIM and ITAM. ITIM stands for immunoreceptor tyrosine-based inhibition motif, while ITAM means immunoreceptor tyrosine-based activation motif. A number of polypeptide receptors bearing one or more ITIM motifs in the cytosolic region of the receptor have been found to be inhibiting, whereas a number of polypeptide receptors bearing one or more ITAM sequences in the cytosolic region have been found to be activating.
The cross-linking of an inhibiting receptor with an activating receptor may result in inhibition of the activating receptor. Traditionally, cross-linking involves the use of three components, where these components are added an incubation medium containing cultured cells, such as cultured T cells or mast cells. Generally, two of these components are antibodies, where each antibody recognizes a different antigen on the cell surface. A third component is often a third independent antibody which recognizes the constant region of the first two antibodies.
A multi-component cross-linking system allows for efficient and controlled studies in conducting research experiments with cultured cells. However, a multi-component cross-linking system is not a practical method for pharmaceutical intervention or drug therapy. One disadvantage is that cross-linking using a three-component system requires four different binding reactions. A second disadvantage is the use of three antibodies to cross-link receptors is therapeutically not feasible.
The present invention addresses these problems by providing one bispecific antibody, which is capable of binding and physiologically affecting an activating receptor and inhibiting receptor on a cell of the immune system.